Heparin administration is the standard antithrombotic therapy indicated for acute venous thrombosis, for prophylaxis in the post surgical (especially orthopedic) and immobile patient, and for flushing of intravenous lines to maintain patency. Approximately 5% (range up to 30%) of patients treated with heparin develop immune-mediated thrombocytopenia (HIT) which may be complicated by either bleeding (as a consequence of decreased platelet count) or arterial and venous thrombosis due to intravascular platelet clumping. This complication occurs in as many as 20% of patients with HIT and may result in serious morbidity and death in about 50% of the cases. Because the diagnosis of HIT poses a serious problem of management, a rapid and reliable determination of HIT is clinically important. Treatment with heparin in the face of HIT results in serious aggravation of the hemostatic complications, hence, heparin therapy in that case should be discontinued. On the other hand, discontinuation of heparin may expose the patient, who requires antithrombotic therapy, to excessive risk of thrombosis since no alternate therapy with immediate and effective antithrombotic capacity is presently available. Moreover, the alternate therapies, such as low-molecular weight heparin or heparinoids, may not be compatible because of potential crossreaction with the anti-heparin antibodies, resulting in further aggregation of HIT. Thus, a rapid and highly reliable affirmative test for HIT is required for correct management decisions. Unfortunately, despite an urgent need thereof, no such test is routinely available to effectively support management decisions and consequently, the initial diagnosis is presently being made on a clinical basis only.
According to the literature the incidence of HIT in patients receiving therapeutic doses of heparin is approximately 5% (range up to 30%). This estimate is somewhat lower in patients receiving heparin in prophylactic doses or in flush solutions (conservative estimate of 2.5%). The incidence of HIT, however, is underestimated. Firstly, because of the lack of a routine test for HIT, only patients who develop significant thrombocytopenia are usually considered for HIT. Secondly, sudden thrombotic events such as myocardial infarction, peripheral arterial thrombosis, and pulmonary emboli are often not recognized as a complication of heparin because of the patient's underlying illnesses. Furthermore, some patients with heparin-induced antibodies have thrombosis in the absence of thrombocytopenia. Thus, all patients receiving heparin who develop thrombosis should be suspected and tested for HIT. In addition, as low-molecular weight heparin is being recommended for prophylaxis for outpatients (a single, subcutaneous dose/day, requiring no monitoring), the incidence of HIT can be expected to increase. Thus, it is reasonable to anticipate that the request for a reliable routine test for HIT will increase significantly.
The method of the present invention obviates the difficulties and limitations that are inherent in the prior art methods that are currently used for detection of HIT. Many of the prior art methods of detecting HIT detect the appearance of antibodies directed against platelets, or the immune complex formed thereof, or a transport function dependent thereon, in the presence of heparin. According to Admiral U.S. Pat. No. 5,466,582 issued Nov. 14, 1995!, "these biological assays are either (a) rather insensitive or rather unreliable or (b) if they are sensitive, lengthy to carry out." For example, a test to diagnose HIT was previously developed based on the finding that a heparin-dependent platelet-aggregating factor appeared in sera of patients with HIT Fratantoni et al. (1975) Blood 45:396!. However, this promising platelet aggregation assay was found to be insensitive and non-specific, giving positive results in some patients without HIT and negative results in some patients with HIT Kelton et al. (1984) J. Lab. Clin. Med. 103:606!. Problems with both sensitivity and specificity seriously compromised the usefulness of this assay for diagnostic use.
A second assay, a serotonin release assay (SRA), was developed from the observation that sera from patients with HIT initiated platelet aggregation and secretion at therapeutic, but not at high, concentrations of heparin. This assay, still regarded as the gold-standard assay for HIT, measures the release of radiolabeled serotonin from platelets at two heparin concentrations, since sera from patients with HIT caused release of serotonin at therapeutic but not at high concentrations of heparin. This test has a high specificity (99%), is very sensitive, and indicates a direct correlation between a positive test result and the clinical likelihood of a patient having HIT Sheridan et al. (1986) Blood 67:27-30!. The serotonin release assay is a more sensitive test of platelet activation than was the aggregation assay. However, certain disadvantages are associated with the serotonin release assay; for example, (a) the use of radiolabeled (.sup.3 H or .sup.4 C) serotonin is required; (b) the assay can be performed only by research or specialized laboratories which carry a license for handling such radioactive materials; and (c) the need to perform the diagnostic serotonin release assay in a licensed laboratory, even for clinical use, denies immediacy for diagnosis of HIT and provides, instead, only retrospective confirmation George et al. (1994) American Society of Hematology, Education Program, Nashville, Tenn., Dec. 2-6!.
An enzyme-linked immunosorbent assay (ELISA) is also currently proposed for use in the detection of HIT by measuring the presence of heparin-Platelet Factor 4-induced antibodies in plasma. Presently, kits for ELISA determination of HIT are currently available only for research purposes (e.g., American Bioproducts Co, Parsippany, N.J.). Although this method measures the presence in test plasma of antibodies that react with heparin-PF4 complexes, this assay is unable to establish a cause-result relationship that relates the presence of antibodies to platelet destruction and thrombocytopenia. The presence per se of antibodies that are immuno-reactive in vitro with heparin-Platelet Factor 4 complexes may or may not be indicative of the presence of HIT. The ELISA method detects only the presence of antibody, irrespective of its pathophysiologic significance. For example, in a related medical case, although up to 36% of patients taking .alpha.-methyldopa develop anti-red cell antibodies, only less than 1% exhibit cell destruction with hemolytic anemia Hematology, 5th ed. (1985) W. J. Williams, E. Beutler, A. J. Erslev and M. A. Lichtman, eds., New York, McGraw-Hill!. Thus, a patient being evaluated for HIT requires that the HIT assay method clearly determine not only the presence of antibodies to a heparin-platelet complex, but also demonstrate that these antibodies are directly relevant to the development of thrombocytopenia.
In addition, assays such as the ELISA are time-consuming, costly and thus are normally not performed for single determinations but samples are accumulated with time to be assayed concurrently as a multiple sample assay.
In contrast to existing prior art methods, the method of the present invention, the flow cytometry assay, is independent of the use of a radiolabeled marker; it provides a high specificity and a sensitivity greater than the serotonin release assay; and in contrast to the ELISA, it is a functional assay for detection of HIT. This method, by reproducing the in vivo pathophysiologic process of HIT, measures specifically platelets that are activated (and consequently destroyed), wherein the activation of platelets is dependent on heparin and test plasma antibodies and wherein the activation of platelets by heparin and test plasma antibodies is diagnostic evidence of in vivo HIT. Additional advantages of the flow cytometry method in comparison to prior art methods are:
(a) the ease of carrying out the flow cytometry assay, such as: PA1 (b) the rapidity and reliability of this assay as a diagnostic assay for HIT which PA1 (c) the ability to determine the effectiveness and compatibility of alternate therapy, such as low molecular weight heparin or heparinoids, which might crossreact with the anti-heparin antibodies and cause HIT.
(i) using readily available reagents, PA2 (ii) employing standard flow cytometric equipment available at any medical center, and PA2 (iii) requiring only standard, and not special, technical expertise; PA2 (i) is available for a therapeutic decision within 1.5 to 2 hours, and PA2 (ii) is cost-effective and can be performed for a single case as well as for multiple cases; and
In comparing the various methods fo diagnosis of HIT, the functional assays are considered clinically superior to immune-detection assays, since the latter detect only the presence of antibodies without establishing a cause-effect relationship with platelet destruction. Therefore, the immune detection assay cannot be directly related to clinical settings. In a recent study of ELISA vs. SRA (serotonin release assay), 22% of samples were found positive with the immune-assay but negative with the functional one Arepally et al. (1995) Am. J. Clin. Pathol. 104:648-654!. As remarked by the authors of the study, "Some patients exposed to heparin may form low-affinity drug-dependent antibodies that . . . may have little correlation with the pathologic activity in vivo". Thus, the authors concluded that "positive ELISA results of this magnitude cannot in and of themselves be considered to provide unequivocal confirmation of the diagnosis of HIT. Performance of the SRA may provide confirmatory evidence in such cases". These remarks may be further highlighted by the observations Kelton et al. (1988) Blood 72:925; Visentin et al. (1994) J. Clin. Invest. 93:81-88! that some patients with positive SRA may not have antibodies detected by immunologic assays. In contrast, the functional flow cytometry assay can effectively provide the necessary information correlating the presence of antibodies with platelet activation.